MicroRNA-574-5p Was Pivotal for TLR9 Signaling Enhanced Tumor Progression via Down-Regulating Checkpoint Suppressor 1 in Human Lung Cancer

Accumulating data suggested that functional expression of Toll-like receptors (TLRs) in tumor cells was involved in tumor progression. Our previous study demonstrated that TLR9 signaling could enhance the tumor progression of human lung cancer cells in vitro and in vivo. We further showed that miR-574-5p was the mostly up-regulated miRNA in human lung cancer cells under TLR9 signaling by miRNA array analysis. Here we characterized the potential role of miRNA-574-5p in enhanced tumor progression induced by TLR9 signaling in human lung cancer. We confirmed that TLR9 signaling effectively elevated the expression of miR-574-5p in human lung cancer cells. Notably, we found that down-regulation of miRNA-574-5p using miR-574-5p inhibitor in vitro or miR-574-5p sponge in vivo significantly abrogated the enhanced tumor progression induced by TLR9 signaling. Further studies showed that miR-574-5p was an important player associated with enhanced tumor progression of human lung cancer cells. Notably, we identified checkpoint suppressor 1 (Ches1) as the dominant direct target for miRNA-574-5p to confer the TLR9 signaling enhanced tumor progression. We revealed that over-expression of Ches1 significantly inhibited the cell cycle entry of human lung cancer cells. Finally, we revealed that the expression of miR-574-5p was positively correlated with TLR9 and reversely correlated with Ches1 in lung cancer patients. Our findings not only facilitated the further understanding of the crosstalk between miRNAs and TLRs in tumor biology, but also provided novel potential candidates for treatment of cancer.     

Depletion of beta-arrestin-2 promotes tumor growth and angiogenesis in a murine model of lung cancer

Arrestins are adaptor/scaffold proteins that complex with activated and phosphorylated G protein-coupled receptor to terminate G protein activation and signal transduction. These complexes also mediate downstream signaling, independently of G protein activation. We have previously shown that beta-arrestin-2 (betaarr2) depletion promotes CXCR2-mediated cellular signaling, including angiogenesis and excisional wound closure. This study was designed to investigate the role of betaarr2 in tumorigenesis using a murine model of lung cancer. To that end, heterotopic murine Lewis lung cancer and tail vein metastasis tumor model systems in betaarr2-deficient mice (betaarr2(-/-)) and control littermates (betaarr2(+/+)) were used. betaarr2(-/-) mice exhibited a significant increase in Lewis lung cancer tumor growth and metastasis relative to betaarr2(+/+) mice. This correlated with decreased number of tumor-infiltrating lymphocytes but with elevated levels of the ELR(+) chemokines (CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2), vascular endothelial growth factor, and microvessel density. NF-kappaB activity was also enhanced in betaarr2(-/-) mice, whereas hypoxia-inducible factor-1alpha expression was decreased. Inhibition of CXCR2 or NF-kappaB reduced tumor growth in both betaarr2(-/-) and betaarr2(+/+) mice. NF-kappaB inhibition also decreased ELR(+) chemokines and vascular endothelial growth factor expression. Altogether, the data suggest that betaarr2 modulates tumorigenesis by regulating inflammation and angiogenesis through activation of CXCR2 and NF-kappaB.     

Effect of beta-carotene on the inhibition of lung metastasis in mice.

Effect of beta-carotene on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice. Simultaneous administration of the compound after tumor induction produced a significant reduction (71.36%) in tumor nodule formation. Increased lung collagen hydroxyproline (22.37 microg/mg protein) in the metastasized lungs of control animals compared to the normal animals (0.95 microg/mg protein) was significantly reduced (4.19 microg/mg protein) in the beta-carotene treated animals. High amount of uronic acid (355.83 microg/100mg tissue ) in the metastasized control animals was significantly reduced (87.87 microg/100 mg tissue) in the animals treated with beta-carotene. Lung hexosamine content also was inhibited significantly in the beta-carotene treated animals (1.58 mg/100 mg lyophilized tissue) compared to the untreated control animals (4.2 mg/100 mg lyophilized tissue). The elevated levels of serum sialic acid and serum gamma glutamyl transpeptidase activity in the untreated control animals was significantly reduced in the animals treated with beta-carotene. Beta carotene treated animals were survived up to 69 days. Histopathology of the lung tissue also correlated with the above parameters and life span of the drug treated animals. Our results reveal the antimetastatic activity of beta-carotene which are abundantly present in green plants, vegetables and fruits.     

Enalapril protects mice from pulmonary hypertension by inhibiting TNF-mediated activation of NF-kappaB and AP-1

The present study was undertaken to investigate the effects of treatment with the angiotensin-converting enzyme (ACE) inhibitor enalapril in a mouse model of pulmonary hypertension induced by bleomycin. Bleomycin-induced lung injury in mice is mediated by enhanced tumor necrosis factor-alpha (TNF) expression in the lung, which determines the murine strain sensitivity to bleomycin, and murine strains are sensitive (C57BL/6) or resistant (BALB/c). Bleomycin induced significant pulmonary hypertension in C57BL/6, but not in BALB/c, mice; average pulmonary arterial pressure (PAP) was 26.4 +/- 2.5 mmHg (P < 0.05) vs. 15.2 +/- 3 mmHg, respectively. Bleomycin treatment induced activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 and enhanced collagen and TNF mRNA expression in the lung of C57BL/6 but not in BALB/c mice. Double TNF receptor-deficient mice (in a C57BL/6 background) that do not activate NF-kappaB or AP-1 in response to bleomycin did not develop bleomycin-induced pulmonary hypertension (PAP 14 +/- 3 mmHg). Treatment of C57BL/6 mice with enalapril significantly (P < 0.05) inhibited the development of pulmonary hypertension after bleomycin exposure. Enalapril treatment inhibited NF-kappaB and AP-1 activation, the enhanced TNF and collagen mRNA expression, and the deposition of collagen in bleomycin-exposed C57BL/6 mice. These results suggest that ACE inhibitor treatment decreases lung injury and the development of pulmonary hypertension in bleomycin-treated mice.     

Effect of Hyperoxia on Retinoid Metabolism and Retinoid Receptor Expression in the Lungs of Newborn Mice

Background

Preterm newborns that receive oxygen therapy often develop bronchopulmonary dysplasia (BPD), which is abnormal lung development characterized by impaired alveologenesis. Oxygen-mediated injury is thought to disrupt normal lung growth and development. However, the mechanism of hyperoxia-induced BPD has not been extensively investigated. We established a neonatal mouse model to investigate the effects of normobaric hyperoxia on retinoid metabolism and retinoid receptor expression.

Methods

Newborn mice were exposed to hyperoxic or normoxic conditions for 15 days. The concentration of retinol and retinyl palmitate in the lung was measured by HPLC to gauge retinoid metabolism. Retinoid receptor mRNA levels were assessed by real-time PCR. Proliferation and retinoid receptor expression in A549 cells were assessed in the presence and absence of exogenous vitamin A.

Results

Hyperoxia significantly reduced the body and lung weight of neonatal mice. Hyperoxia also downregulated expression of RARα, RARγ, and RXRγ in the lungs of neonatal mice. In vitro, hyperoxia inhibited proliferation and expression of retinoid receptors in A549 cells.

Conclusion

Hyperoxia disrupted retinoid receptor expression in neonatal mice.     

Chitinase 1 Is a Biomarker for and Therapeutic Target in Scleroderma-Associated Interstitial Lung Disease That Augments TGF-β1 Signaling

Interstitial lung disease (ILD) with pulmonary fibrosis is an important manifestation in systemic sclerosis (SSc, scleroderma) where it portends a poor prognosis. However, biomarkers that predict the development and or severity of SSc-ILD have not been validated, and the pathogenetic mechanisms that engender this pulmonary response are poorly understood. In this study, we demonstrate in two different patient cohorts that the levels of chitotriosidase (Chit1) bioactivity and protein are significantly increased in the circulation and lungs of SSc patients compared with demographically matched controls. We also demonstrate that, compared with patients without lung involvement, patients with ILD show high levels of circulating Chit1 activity that correlate with disease severity. Murine modeling shows that in comparison with wild-type mice, bleomycin-induced pulmonary fibrosis was significantly reduced in Chit1(-/-) mice and significantly enhanced in lungs from Chit1 overexpressing transgenic animals. In vitro studies also demonstrated that Chit1 interacts with TGF-β1 to augment fibroblast TGF-β receptors 1 and 2 expression and TGF-β-induced Smad and MAPK/ERK activation. These studies indicate that Chit1 is potential biomarker for ILD in SSc and a therapeutic target in SSc-associated lung fibrosis and demonstrate that Chit1 augments TGF-β1 effects by increasing receptor expression and canonical and noncanonical TGF-β1 signaling.     

Investigation into the cancer protective effect of flaxseed in Tg.NK (MMTV/c-neu) mice, a murine mammary tumor model

The aim of the present study was to investigate whether low flaxseed doses relevant to human dietary exposure can prevent mammary tumors in transgenic Tg.NK mice, a model of breast cancer. Animals were exposed to flaxseed through the diet at human relevant levels. Tumor-related parameters and tumor development were evaluated. Hepatic cytochrome P450 and glutathione S-transferase activities were significantly reduced in animals receiving low flaxseed doses. An incidence of palpable tumors before sacrifice, a number of tumors per mouse, and a number of large tumors (>6 mm diameter) at necropsy were statistically significantly lower in the high flaxseed group compared to controls, suggesting a beneficial effect on tumor progression of small dietary doses of flaxseed. However, the number of tumor-bearing mice and multiplicity of tumors at necropsy were not statistically significantly lower compared to the controls. Thus, the effect of small dietary doses of flaxseed on mammary tumor development in Tg.NK mice remains to be established.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-011-0214-1) contains supplementary material, which is available to authorized users.     

Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor

Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.     

Biogenesis of retinoic acid from beta-carotene. Differences between the metabolism of beta-carotene and retinal

The ability of beta-carotene to serve as precursor to retinoic acid was examined in vitro with cytosol prepared from rat tissues. The rate of retinoic acid synthesis from 10 microM beta-carotene ranged from 120 to 224 pmol/h/mg of protein with intestinal cytosol, and from 344 to 488 pmol/h/mg of protein with cytosols prepared from kidney, lung, testes, and liver. Retinol generated during beta-carotene metabolism was not the major substrate for retinoic acid synthesis. At low substrate concentrations (2.5 microM), the rates of retinoic acid synthesis in intestinal cytosol from beta-carotene or retinol were equivalent, and at higher concentrations (10 microM) the rates of retinoic acid synthesis from beta-carotene or retinol in intestine, testes, lung, and kidney were comparable. Thus, beta-carotene metabolism may be an important source of retinoic acid in retinoid target tissues, particularly in species such as humans that are capable of accumulating high concentrations of tissue carotenoids. Retinal, considered an initial retinoid product of beta-carotene metabolism, was not detected as a product of beta-carotene metabolism in vitro. A ratio of retinol and retinoic acid different from that observed during beta-carotene metabolism in vitro was observed with incubations of retinal under identical conditions. These data indicated that beta-carotene metabolism is not merely a simple process of producing retinal and releasing it into solution to be metabolized independently.     

Critical modifier role of membrane-cystic fibrosis transmembrane conductance regulator-dependent ceramide signaling in lung injury and emphysema

Ceramide accumulation mediates the pathogenesis of chronic obstructive lung diseases. Although an association between lack of cystic fibrosis transmembrane conductance regulator (CFTR) and ceramide accumulation has been described, it is unclear how membrane-CFTR may modulate ceramide signaling in lung injury and emphysema. Cftr(+/+) and Cftr(-/-) mice and cells were used to evaluate the CFTR-dependent ceramide signaling in lung injury. Lung tissue from control and chronic obstructive pulmonary disease patients was used to verify the role of CFTR-dependent ceramide signaling in pathogenesis of chronic emphysema. Our data reveal that CFTR expression inversely correlates with severity of emphysema and ceramide accumulation in chronic obstructive pulmonary disease subjects compared with control subjects. We found that chemical inhibition of de novo ceramide synthesis controls Pseudomonas aeruginosa-LPS-induced lung injury in Cftr(+/+) mice, whereas its efficacy was significantly lower in Cftr(-/-) mice, indicating that membrane-CFTR is required for controlling lipid-raft ceramide levels. Inhibition of membrane-ceramide release showed enhanced protective effect in controlling P. aeruginosa-LPS-induced lung injury in Cftr(-/-) mice compared with that in Cftr(+/+) mice, confirming our observation that CFTR regulates lipid-raft ceramide levels and signaling. Our results indicate that inhibition of de novo ceramide synthesis may be effective in disease states with low CFTR expression like emphysema and chronic lung injury but not in complete absence of lipid-raft CFTR as in ΔF508-cystic fibrosis. In contrast, inhibiting membrane-ceramide release has the potential of a more effective drug candidate for ΔF508-cystic fibrosis but may not be effectual in treating lung injury and emphysema. Our data demonstrate the critical role of membrane-localized CFTR in regulating ceramide accumulation and inflammatory signaling in lung injury and emphysema.     

Suppression of insulin receptor substrate 1 (IRS-1) promotes mammary tumor metastasis

The insulin receptor substrate (IRS) proteins are cytoplasmic adaptors that organize signaling complexes downstream of activated cell surface receptors. Here, we show that IRS-1 and IRS-2, despite significant homology, play critical yet distinct functions in breast cancer, and we identify specific signaling pathways that are influenced by IRS-1 using the polyoma virus middle-T (PyV-MT) transgenic mouse model of mammary carcinoma and Irs-1 null (Irs1(-/-)) mice. The absence of Irs-1 expression enhanced metastatic spread significantly without a significant effect on primary tumor growth. Orthotopic transplant studies revealed that the increased metastatic potential of Irs1-deficient tumor cells is cell autonomous. Mammary tumors that developed in PyV-MT::Irs1(-/-) mice exhibited elevated Irs-2 function and enhanced phosphatidylinositol 3-kinase/Akt/mTor activity, suggesting that one mechanism by which Irs-1 impedes metastasis is to suppress Irs-2-dependent signaling. In support of this mechanism, reduction of Irs-2 expression in Irs1(-/-) tumor cells restored mTor signaling to wild-type levels. PyV-MT::Irs1(-/-) tumors also exhibited a significant increase in vascular endothelial growth factor expression and microvessel density, which could facilitate their dissemination. The significance of our findings for human breast cancer is heightened by our observation that Irs-1 is inactivated in wild-type, metastatic mammary tumors by serine phosphorylation. Collectively, our findings reveal that inactivation of IRS-1 enhances breast cancer metastasis and support the novel hypothesis that IRS-1 has metastasis suppressor functions for breast cancer.     

Microenvironment-derived IL-1 and IL-17 interact in the control of lung metastasis

Inflammatory cytokines modulate immune responses in the tumor microenvironment during progression/metastasis. In this study, we have assessed the role of IL-1 and IL-17 in the control of antitumor immunity versus progression in a model of experimental lung metastasis, using 3LL and B16 epithelial tumor cells. The absence of IL-1 signaling or its excess in the lung microenvironment (in IL-1β and IL-1R antagonist knockout [KO] mice, respectively) resulted in a poor prognosis and reduced T cell activity, compared with WT mice. In IL-1β KO mice, enhanced T regulatory cell development/function, due to a favorable in situ cytokine network and impairment in APC maturation, resulted in suppressed antitumor immunity, whereas in IL-1R antagonist KO mice, enhanced accumulation and activity of myeloid-derived suppressor cells were found. Reduced tumor progression along with improved T cell function was found in IL-17 KO mice, compared with WT mice. In the microenvironment of lung tumors, IL-1 induces IL-17 through recruitment of γ/δ T cells and their activation for IL-17 production, with no involvement of Th17 cells. These interactions were specific to the microenvironment of lung tumors, as in intrafootpad tumors in IL-1/IL-17 KO mice, different patterns of invasiveness were observed and no IL-17 could be locally detected. The results highlight the critical and unique role of IL-1, and cytokines induced by it such as IL-17, in determining the balance between inflammation and antitumor immunity in specific tumor microenvironments. Also, we suggest that intervention in IL-1/IL-17 production could be therapeutically used to tilt this balance toward enhanced antitumor immunity.     

Flavonoids suppresses the enhancing effect of beta-carotene on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A549 cells

This study investigated the individual and combined effects of beta-carotene with a common flavonoid (naringin, quercetin or rutin) on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-related carcinogen in human. A human lung cancer cell line, A549, was pre-incubated with beta-carotene, a flavonoid, or both for 1h followed by incubation with NNK for 4 h. Then, we determined DNA strand breaks and the level of 7-methylguanine (7-mGua), a product of NNK metabolism by cytochrome P450 (CYP). We showed that beta-carotene at 20 microM significantly enhanced NNK-induced DNA strand breaks and 7-mGua levels by 90% (p < 0.05) and 70% (p < 0.05), respectively, and that the effect of beta-carotene was associated with an increased metabolism of NNK by CYP because the concomitant addition of 1-aminobenzotriazole, a CYP inhibitor, with beta-carotene to cells strongly inhibited NNK-induced DNA strand breaks. In contrast to beta-carotene, incubation of cells with naringin, quercetin or rutin added at 23 microM led to significant inhibition of NNK-induced DNA strand breaks, and the effect was in the order of quercetin > naringin > rutin. However, these flavonoids did not significantly affect the level of 7-mGua induced by NNK. Co-incubation of beta-carotene with any of these flavonoids significantly inhibited the enhancing effect of beta-carotene on NNK-induced DNA strand breaks; the effects of flavonoids were dose-dependent and were also in the order of quercetin > naringin > rutin. Co-incubation of beta-carotene with any of these flavonoids also significantly inhibited the loss of beta-carotene incorporated into the cells, and the effects of the flavonoids were also in the order of quercetin > naringin > rutin. The protective effects of these flavonoids may be attributed to their antioxidant activities because they significantly decreased intracellular ROS, and the effects were also in the order of quercetin > naringin > rutin. These in vitro results suggest that a combination of beta-carotene with naringin, rutin, or quercetin may increase the safety of beta-carotene.     

Role of PAR2 in murine pulmonary pseudomonal infection

Proteinases can influence lung inflammation by various mechanisms, including via cleavage and activation of protease-activated receptors (PAR) such as PAR2. In addition, proteinases such as neutrophil and/or Pseudomonas-derived elastase can disarm PAR2 resulting in loss of PAR2 signaling. Currently, the role of PAR2 in host defense against bacterial infection is not known. Using a murine model of acute Pseudomonas aeruginosa pneumonia, we examined differences in the pulmonary inflammatory response between wild-type and PAR2(-/-) mice. Compared with wild-type mice, PAR2(-/-) mice displayed more severe lung inflammation and injury in response to P. aeruginosa infection as indicated by higher bronchoalveolar lavage fluid neutrophil numbers, protein concentration, and TNF-alpha levels. By contrast, IFN-gamma levels were markedly reduced in PAR2(-/-) compared with wild-type mice. Importantly, clearance of P. aeruginosa was diminished in PAR2(-/-) mice. In vitro testing revealed that PAR2(-/-) neutrophils killed significantly less bacteria than wild-type murine neutrophils. Further, both neutrophils and macrophages from PAR2(-/-) mice displayed significantly reduced phagocytic efficiency compared with wild-type phagocytes. Stimulation of PAR2 on macrophages using a PAR2-activating peptide resulted in enhanced phagocytosis directly implicating PAR2 signaling in the phagocytic process. We conclude that genetic deletion of PAR2 is associated with decreased clearance of P. aeruginosa. Our data suggest that a deficiency in IFN-gamma production and impaired bacterial phagocytosis are two potential mechanisms responsible for this defect.     

Pro-carcinogenic activity of beta-carotene, a putative systemic photoprotectant.

Beta-carotene is a strong singlet oxygen quencher and antioxidant. Epidemiologic studies have implied that an above average intake of the carotenoid might reduce cancer risks. Earlier studies found that the carotenoid, when added to commercial closed-formula rodent diets, provided significant photoprotection against UV-carcinogenesis in mice. Clinical intervention trials found that beta-carotene supplementation evoked no change in incidence of nonmelanoma skin cancer. However, when smokers were supplemented with the carotenoid a significant increase in lung cancer resulted. Recently, employing a beta-carotene supplemented semi-defined diet, not only was no photoprotective effect found, but significant exacerbation of UV-carcinogenesis occurred. Earlier, a mechanism, based upon redox potential of interacting antioxidants, was proposed in which beta-carotene participated with vitamins E and C to efficiently repair oxy radicals and, thus, thought to provide photoprotection. In this schema, alpha-tocopherol would first intercept an oxy radical. In terminating the radical-propagating reaction, the tocopherol radical cation is formed which, in turn, is repaired by beta-carotene to form the carotenoid radical cation. This radical is repaired by ascorbic acid (vitamin C). As the carotenoid radical cation is a strongly oxidizing radical, unrepaired it could contribute to the exacerbating effect on UV-carcinogenesis. Thus, vitamin C levels could influence the levels of the pro-oxidant carotenoid radical cation. However, when hairless mice were fed beta-carotene supplemented semi-defined diet with varying levels of vitamin C (0-5590 mg kg(-1) diet) no effect on UV-carcinogenesis was observed. Lowering alpha-tocopherol levels did result in further increase of beta-carotene exacerbation, suggesting beta-carotene and alpha-tocopherol interaction. It was concluded that the non-injurious or protective effect of beta-carotene found in the closed-formula rations might depend on interaction with other dietary factors that are absent in the semi-defined diet. At present, beta-carotene use as a dietary supplement for photoprotection should be approached cautiously.     

Altered tumor biology and tumorigenesis in irradiated and chemical carcinogen-treated single and combined connexin32/p27Kip1-deficient mice

Connexin32 knockout mice (Cx32-KO) exhibit increased chemical and radiation-induced liver and lung tumorigenesis. This increased tumor incidence is associated with altered tumor biology including enhanced tumor progression and an increased percent of MAPK-active tumors. Likewise, mice lacking the tumor suppressor/cell cycle regulator p27Kip1 exhibit increased tumorigenesis in a variety of tissues following chemical and radiation induction. Interestingly, in a double-deficient mouse model (DKO), additional loss of p27Kip1 in a Cx32-KO background results in attenuation of liver and lung tumorigenesis as well as MAPK activation profiles, suggesting pathway interaction. While these mouse strains exhibit altered liver and lung tumor susceptibility following both chemical (DEN) and radiation (X-ray) induction protocols, comparisons of the resulting tumor incidence, multiplicity, tumor progression, and MAPK activation in response to these two distinct carcinogens underscores the separate influence of each individual gene on both tumor formation and activation of specific oncogenic pathways. Furthermore, these studies demonstrate that different carcinogens interact disparately with Cx32/p27Kip1 genotypic backgrounds in situ resulting in varied tumorigenic response.